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CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The costimulatory domains incorporated into second-generation and third-generation chimeric antigen receptors (CARs) strongly influence CAR-T-cell function. Here, we explored second-generation and third-generation CARs harboring the signaling domain of the CD40 receptor as a new costimulatory element in comparison with similar CARs carrying the 4-1BB domain. In CARs of both generations, CD40 was more potent than 4-1BB in triggering the NF-κB signaling pathway. In human T cells from 2 donors, CD40 was comparable to 4-1BB in upregulating costimulatory and activation markers, inducing proinflammatory cytokine secretion and mediating target cell killing. Interestingly, differences in the response pattern of T cells from the 2 donors with respect to CD40 and 4-1BB were evident. We conclude that in human T cells, the CD40 signaling domain is a potent costimulatory element in both second-generation and third-generation CARs., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cell-cell interactions involving specific membrane proteins are critical triggers in cellular development. Ex vivo strategies to mimic these effects currently use soluble proteins or (recombinant) presenter cells, albeit with mixed results. A promising alternative are bacterial magnetosomes, which can be selectively transformed into cell-free membrane-protein presenters by genetic engineering. In this study, the human CD40 Ligand (CD40L), a key ligand for B cell activation, is expressed on the particle surface. Functionality is demonstrated on sensor cells expressing the human CD40 receptor. Binding of CD40L magnetosomes to these cells triggers a signaling cascade leading to the secretion of embryonic alkaline phosphatase. Concomitantly, the CD40-CD40L interaction is strong enough to allow cell recovery by magnetic sorting. Overall, this study demonstrates the potential of magnetosomes as promising cell-free tools for cellular biotechnology, based on the display of membrane-bound target molecules, thereby creating a biomimetic interaction., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The antagonistic anti-CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo- and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N-terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross-blocking anti-CD40 antibodies 3A8 and 5D12. All four 2C10-binding residues defined by mutagenesis clustered near the membrane-distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications., Results: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies., Conclusions: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

We report the design, synthesis, and testing of novel small-molecule compounds targeting the CD40⁻CD154 (CD40L) costimulatory interaction for immunomodulatory purposes. This protein-protein interaction (PPI) is a TNF-superfamily (TNFSF) costimulatory interaction that is an important therapeutic target since it plays crucial roles in the activation of T cell responses, and there is resurgent interest in its modulation with several biologics in development. However, this interaction, just as all other PPIs, is difficult to target by small molecules. Following up on our previous work, we have now identified novel compounds such as DRI-C21091 or DRI-C21095 that show activity (IC 50 ) in the high nanomolar to low micromolar range in the binding inhibition assay and more than thirty-fold selectivity versus other TNFSF PPIs including OX40⁻OX40L, BAFFR-BAFF, and TNF-R1-TNFα. Protein thermal shift (differential scanning fluorimetry) assays indicate CD154 and not CD40 as the binding partner. Activity has also been confirmed in cell assays and in a mouse model (alloantigen-induced T cell expansion in a draining lymph node). Our results expand the chemical space of identified small-molecule CD40⁻CD154 costimulatory inhibitors and provide lead structures that have the potential to be developed as orally bioavailable immunomodulatory therapeutics that are safer and less immunogenic than corresponding biologics.

Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis B virus (HBV) infection is a major public health problem and immune tolerance is responsible for persistent HBV infection. HBV therapeutic vaccines targeting HBV e antigen (HBeAg) may have an excellent effect in overcoming HBV immune tolerance. Thus, there is urgency for designing therapeutic vaccine candidates that target HBeAg. In this research, we fused the C (472-507) gene sequence of HBV with the extracellular domain of human CD40 ligand sequence and ligated this fused sequence into the pEGFP-N1 vector to construct the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L. Then, the dendritic cells (DCs) generated from human peripheral blood were transfected with this recombinant plasmid. After this, the phenotype and function of DCs were assessed. Compared with the three control groups of pEGFP-N1-C (472-507), pEGFP-N1 and phosphate buffered saline (PBS), we found that DCs transfected with the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L enhanced the expression of costimulatory molecules (CD80, CD86 and HLA-DR) and secretion of cytokine IL-12p70. Furthermore, the capacity of inducing the proliferation of allogeneic lymphocytes was also improved. Our study validated that transfecting DCs with recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L could activate DCs and enhance their functions. Therefore, C (472-507)-ecdCD40L fusion sequence may be a promising vaccine candidate for chronic hepatitis B therapythat targets HBeAg.

CD40 is known as "master switch" in immune response to pathogen infection in mammals. However, limited information of CD40 is known in lower vertebrates. In this study, a novel CD40 homolog (Ls-CD40) was cloned and characterized from humphead snapper, Lutjanus sanguineus. The Ls-CD40 cDNA composed of 2073 bp with a 69 bp of 5'-UTR, a 1020 bp of 3'-UTR and an open reading frame (ORF) of 984 bp, encoding 327 amino acid residues. Sequence analysis showed that Ls-CD40 contained a single peptide, a transmembrane domain and four cysteine-rich domains. The deduced amino acid sequence of Ls-CD40 shared 40%-53% identities with other known fish CD40. The qRT-PCR showed that Ls-CD40 gene expressed in all examined tissues with the most abundant in spleen and lowest level in intestine. After V. harveyi and poly I:C stimulation, the expression of CD40 were significantly induced in spleen. Moreover, Ls-CD40 could interact with Ls-TRAF3 in vitro. These data indicate that Ls-CD40 might play a regulatory role in immune response of L. sanguineus., (Copyright © 2017. Published by Elsevier Ltd.)

The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: CD40, also called Bp50, is a novel member of the TNF receptor superfamily. Based on its important role in multiple physiological and pathological processes, the CD40 signaling pathway has become a vital target for treating transplantation, autoimmune diseases and cancers. This study generated a protein fragment that disrupts this signaling pathway., Results: A DNA fragment encoding the extracellular domain of CD40 (CD40-N) has been codon-optimized and cloned into pPIC9K to create a Pichia pastoris expression and secretion strain. SDS-PAGE and Western blotting assays using the culture media from methanol-induced expression strains showed that recombinant CD40-N, a 27 kDa glycosylated protein, was secreted into the culture broth. The recombinant protein was purified to more than 90 % using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange. Finally, 120 mg of the protein was obtained at a relatively high purity from 3 l supernatant. Binding assay (ITC200 assay) shown the direct interaction of CD40-N and CD40 agonist antibody (G28-5). The bioactivity of recombinant CD40-N was confirmed by its ability to disrupt non-canonical NF-κB signaling activated by CD40 agonist antibody or CD40 ligand and to inhibit ant-CD40 agonist antibody-induced TNF-alpha expression in BJAB cells in vitro. In addition, our data indicate that the protein has curative potential in treating dextran sulfate sodium (DSS)-induced colitis in vivo., Conclusions: The results show that the experimental procedure we have developed using P. pastoris can be used to produce large amounts of active CD40-N for research and industrial purposes. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis.

CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cysteine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD40, as a single receptor that binds CD154 (CD40-ligand or CD40L), regulates counteractive effector functions such as production of pro- and anti-inflammatory cytokines. Therefore, we examined whether such dual messages are encrypted in CD40L. As such message encryption was never investigated, we hypothesized that mutation of certain amino acid residues should in principle enhance pro-inflammatory cytokine production whereas mutation of some others would enhance anti-inflammatory cytokine secretion. We mutated six such residues, which were previously showed to participate in CD40L function. Here, we report that the mutant CD154 129E→V was superior to the wild-type CD154 in killing of Leishmania donovani, induction of inducible nitric oxide synthase (iNOS) and production of IL-12 and relative phosphorylation of p38MAPK and ERK-1/2 in PBMC-derived macrophages. By contrast, 128S→V promoted L. donovani survival, reducing iNOS, but increasing IL-10 expression and predominant ERK-1/2 phosphorylation. The mutant 144G→V did not have significant effects. Other mutants (142E→V, 143K→A, 145Y→F) mimicked the wild-type CD154. Molecular dynamics simulation suggested that these mutations induced differential conformational changes in the CD40-CD154 complex. Therefore, assortment of the contrasting messages encrypted in a given ligand performing counteractive functions presents a novel fundamental biological principle that can be used for devising various therapies., (Copyright © 2015. Published by Elsevier Inc.)

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013., Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse., Results: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice., Conclusions: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer., (©2014 American Association for Cancer Research.)

CD20 is an attractive therapeutic target given the success of its monoclonal antibody, Rituximab, in the treatment of B-cell malignancies and B-cell-mediated autoimmune diseases. Treatment with Rituximab causes a rapid depletion of B cells and a decrease in disease symptoms. Despite the clinical efficiency of Rituximab, its mechanism of action is not completely understood. In this study, we aimed at further investigating the Rituximab-induced cell death and the factors affecting such responses. Our results indicate that Rituximab-induced cell death depends on the nature of the cells and levels of CD20 expression on the cell surface. Coexpression of CD20 with CD40, a member of the TNF receptor family that is known to be physically associated with CD20 on the cell surface, enhances the apoptotic response induced by Rituximab. Inhibiting the formation of CD40 disulfide-bound-homodimers, a process required for some CD40 signaling, further enhances Rituximab-induced cell death. Cell death induced by anti-CD40 mAb is also upregulated by the presence of CD20, suggesting a bidirectional influence of the CD20/CD40 association. Moreover, treating cells with both anti-CD20 and anti-CD40 antibodies improves the cell death response induced by a single-agent treatment. These results highlight the role of the CD20/CD40 association in triggering B-cell depletion and may pave the way for an alternative more efficient therapeutic strategy in treating B-cell-mediated disorders., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There has been increasing interest in the role played by B cells and their associated antibody in the immune response to an allograft, driven by the need to undertake antibody-incompatible transplantation and evidence suggesting that B cells play a role in acute T-cell-mediated rejection and in acute and chronic antibody-mediated rejection. This review focuses on the molecular events, both activating and inhibitory, which control B-cell activation, and considers how this information might inform therapeutic strategies. Potential targets include the BAFF (B-cell-activating factor belonging to the tumour necrosis factor family) and CD40-CD40L pathways and inhibitory molecules, such as CD22 and FcγRIIB. B cells can also play an immunomodulatory role via interleukin (IL)10 production and may contribute to transplant tolerance. The expansion of allograft-specific IL10-producing B cells may be an additional therapeutic goal. Thus, the treatment paradigm required in transplantation has shifted from that of simple B-cell depletion, to that of a more subtle, differential manipulation of different B-cell subsets., (© 2013 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5β1 or αMβ2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.

Objective: To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity., Methods: Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA., Results: The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity., Conclusion: The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a K(D) of 0.6 μM. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.

The CD40L/CD40 signalling pathway is critically involved in the final stage of the maturation of DCs. This paper reports the identification and functional characterization of CD40L and CD40 from Atlantic salmon (Salmo salar). Salmon CD40L is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, while CD40 is a type I membrane-bound receptor with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The salmon CD40L and CD40 were widely expressed, particularly in immune tissues, and while CD40L expression was induced by in vitro stimulation of HKLs with PHA and ConA, CpG increased CD40 expression. A CD40L construct was overexpressed in the CHSE-214 cell line and co-cultivation of the CD40L-CHSE transfectants with HKL induced a rapid and long-lasting upregulation of important costimulatory molecules like CD40, CD83, B7-H1 and the cytokines IL-12p40, IL-10, IL-1β and IFNs, which all are involved in T-helper cell responses. Furthermore, the CD40L transfected cells increased the percentage of HKLs expressing surface MHCIIβ but unlike other APC maturation stimuli, like CpG, they did not reduce the capacity to internalise antigen. Our results provide the first evidence for the existence of a functional CD40L mediated costimulatory pathway in Atlantic salmon., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to "mark" inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules.

CD40 has an important role in T cell-B cell interaction which rescues B lymphocytes from undergoing apoptosis. However, various studies have demonstrated that CD40 can also play a direct role in the induction of specific cell death and thus in the inhibition of tumour cell proliferation. Our previous studies showed that CD40-mediated cell death was independent of caspases and required no de novo protein synthesis. Knowing that CD40 signaling is mediated by its association with several intracellular effectors, including members of TNFR-associated factors (TRAFs) family, the goal of the present study is to investigate the mechanisms involved in the induction of cell death by CD40. Our data reveals that CD40-mediated cell death required lysosomal membrane permeabilization and the subsequent cathepsin B release. In addition, CD40 homodimer formation, a phenomenon known to be necessary for some CD40-mediated signals, was shown to negatively regulate cell death induced by CD40. Moreover, using HEK293 cells ectopically expressing CD40 deficient in TRAF binding, we showed that CD40-mediated apoptosis occurred in the absence of TRAF2 and TRAF3 association, but was significantly reduced when CD40 was deficient in its TRAF6 binding. Therefore, by outlining the role of lysosomal pathways and intracellular effectors, namely TRAF6 in CD40-mediated cell death, our study identifies new targets for anti-cancer therapy., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Interaction of CD40 with CD154 leads to recruitment of both molecules into lipid rafts, resulting in bi-directional cell activation. The precise mechanism by which CD154 is translocated into lipid rafts and its impact on CD154 signaling remain largely unknown. Our aim is to identify the domain of CD154 facilitating its association to lipid rafts and the impact of such association on signaling events and cytokine production. Thus, we generated Jurkat cell lines expressing truncated CD154 lacking the cytoplasmic domain or chimeric CD154 in which the transmembrane domain was replaced by that of transferrin receptor I, known to be excluded from lipid rafts. Our results show that cell stimulation with soluble CD40 leads to the association of CD154 wild-type and CD154-truncated, but not CD154-chimera, with lipid rafts. This is correlated with failure of CD154-chimera to activate Akt and p38 MAP kinases, known effectors of CD154 signaling. We also found that CD154-chimera lost the ability to promote IL-2 production upon T cell stimulation with anti-CD3/CD28 and soluble CD40. These results demonstrate the implication of the transmembrane domain of CD154 in lipid raft association, and that this association is necessary for CD154-mediated Akt and p38 activation with consequent enhancement of IL-2 production.

Hemodynamic forces are important effectors of endothelial cell phenotype and function. Because CD40-CD154 interactions between endothelial cells and mononuclear leukocytes or activated platelets play an important role in vascular dysfunction, we investigated the effects of cyclic stretch on CD40 expression in human cultured endothelial cells. Short-term stretch transiently up-regulated CD40 expression while long-term stretch resulted in a distinct decline in CD40 protein which was prevented by inhibition of the 20S proteasome or scavenging of peroxynitrite. Tyrosine nitration of CD40 also occurred under static conditions on addition of authentic peroxynitrite, and according to mass spectrometry analysis Tyr-82 but not Tyr-31 was its target in the native protein. Immunofluorescence analysis of endothelial cells transduced with a control or Tyr-82 to Ala mutated AAV9-CD40-eGFP expression construct confirmed a peroxynitrite-dependent redistribution of the protein from the cell membrane to the cytoplasm, which was prevented by methyl-β-cyclodextrin. Moreover, CD154-stimulated IL-12p40 and E-selectin expression markedly decreased after exposure to authentic peroxynitrite or cyclic stretch, respectively. Coimmunoprecipitation demonstrated a decreased binding of TRAF2 and TRAF6 to the CD40 protein after tyrosine nitration. Through this posttranslational oxidative modification of an important costimulatory molecule, endothelial cells are able to quickly adapt to unfavorable hemodynamics and maintain their anti-inflammatory phenotype.

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser(132) loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.

Antibody modified magnetic polymeric microspheres have potential biomedical application. In this paper, anti-CD40 antibody modified magnetic poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL, PCEC) microspheres were prepared. First, PCL-PEG-PCL triblock copolymer was synthesized by ring-opening polymerization, followed by reaction with succinic anhydride, creating carboxylated PCL-PEG-PCL copolymer. Then, magnetite nanoparticles were encapsulated into carboxylated PCL-PEG-PCL microspheres, forming magnetic PCL-PEG-PCL microspheres with carboxyl group on their surface. Catalyzed by EDC/NHS, the anti-CD40 antibody was linked to these magnetic PCL-PEG-PCL microspheres, thus forming anti-CD40 modified PCL-PEG-PCL microspheres. These anti-CD40 antibody modified magnetic PCL-PEG-PCL microspheres may have potential application in cell separation.

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.

CD40/CD40 ligand (CD40L) cross-talk plays a key role in B-cell terminal maturation in the germinal centers. Genetic defects affecting CD40 cause a rare form of hyper-immunoglobulin M (IgM) syndrome, a disorder characterized by low or absent serum IgG and IgA, associated with recurrent infections. We previously reported on a few patients with homozygous CD40 mutations resulting in lack or severe reduction of CD40 cell surface expression. Here we characterize the 3 CD40 mutants due to missense mutations or small in-frame deletions, and show that the mutated proteins are synthesized but retained in the endoplasmic reticulum (ER), likely due to protein misfolding. Interestingly, the intracellular behavior and fate differ significantly among the mutants: progressive accumulation of the P2 mutant causes endoplasmic reticulum stress and the activation of an unfolded protein response; the mutant P4 is rather efficiently disposed by the ER-associated degradation pathway, while the P5 mutant partially negotiates transport to the plasma membrane, and is competent for CD40L binding. Interestingly, this latter mutant activates downstream signaling elements when overexpressed in transfected cells. These results give new important insights into the molecular pathogenesis of HIGM disease, and suggest that CD40 deficiency can also be regarded as an ER-storage disease.

The CD40-CD154 dyad is an intensely studied field as is glycosylation status and both impact immunological functions and autoimmune conditions. CD40 has several isoforms, is modified by glycosylation, and trimerizes to form the functional receptor. We described a CD4(+)CD40(+) T cell (Th40) subset which is expanded in autoimmunity and is necessary and sufficient in transferring type 1 diabetes. Glycosylation impacts immunological events and T cells from autoimmune mouse strains express 30-40% less GlcNAc-branched N-glycans than T cells from non-autoimmune strains, a decrease known to activate T cells. Here we demonstrate that several CD40 receptor constellations exist on CD4 T cells. However, rather than containing different isoforms of CD40 they contain different glycoforms of isoform I. The glycoform profile is dependent on availability of CD154 and autoimmune NOD mice express a high level of a less glycosylated form. Interestingly, CD40 stimulation induces some CD40 receptor constellations that contain TNF-receptors 1 and 2 and targeting of those alters CD40 signaling outcomes in NOD Th40 cells. CD40-stimulation in vivo of non-autoimmune BALB/c mice expands the Th40 population and alters the CD40 glycoform profile of those cells to appear more like that of autoimmune prone NOD mice. Further understanding the dynamics and composition of the different CD40 receptor constellations will provide important insights into treatment options in autoimmunity., ((c) 2010 Elsevier Ltd. All rights reserved.)

X-linked Hyper-IgM syndrome (XHIM) is caused by mutations of CD154 gene also known as CD40 ligand (CD40L). CD40L is expressed in activated T cells and interacts with CD40 receptor expressed on B lymphocytes and dendritic cells. Affected patients present cellular and humoral immune defects, with infections by intracellular, opportunistic and extracellular pathogens. In the present study, we investigate molecular defects involved in the XHIM in five patients and identified five distinct CD40L mutations, three of which had not been previously described. P1 harboured a novel p.L193P mutation, which abolished the expression of CD40L. P2 had a frameshift deletion in exon 3 (p.E108fsX19), which also decreased the protein expression. P3 demonstrated p.E54X change in exon 2. P4 harboured the p.Q186X change in the exon 5. P5 demonstrated p. E142X change in exon 5. Mutations in P3, P4 and P5 all led to the production of premature CD40L protein. Two of the five genetically defined patients received umbilical cord blood stem cell transplantation from unrelated donor and achieved clinical remission, and the expression of CD40L on the peripheral blood mononuclear cells restored. These mutations reflect the heterogeneity of CD40L gene, indicating the need for accurate and reliable molecular testing in patients suspected of XHIM.

It is becoming increasingly clear that small molecules can often act as effective protein-protein interaction (PPI) inhibitors, an area of increasing interest for its many possible therapeutic applications. We have identified several organic dyes and related small molecules that (i) concentration-dependently inhibit the important CD40-CD154 costimulatory interaction with activities in the low micromolar (microM) range, (ii) show selectivity toward this particular PPI, (iii) seem to bind on the surface of CD154, and (iv) concentration-dependently inhibit the CD154-induced B cell proliferation. They were identified through an iterative activity screening/structural similarity search procedure starting with suramin as lead, and the best smaller compounds, the main focus of the present work, achieved an almost 3-fold increase in ligand efficiency (DeltaG(0)/nonhydrogen atom = 0.8 kJ/N(nHa)) approaching the average of known promising small-molecule PPI inhibitors (approximately 1.0 kJ/N(nHa)). Since CD154 is a member of the tumor necrosis factor (TNF) superfamily of cell surface interaction molecules, inhibitory activities on the TNF-R1-TNF-alpha interactions were also determined to test for specificity, and the compounds selected here all showed more than 30-fold selectivity toward the CD40-CD154 interaction. Because of their easy availability in various structural scaffolds and because of their good protein-binding ability, often explored for tissue-specific staining and other purposes, such organic dyes can provide a valuable addition to the chemical space searched to identify small molecule PPI inhibitors in general., ((c) 2009 John Wiley & Sons, Ltd.)

The EBV protein, latent membrane protein 1 (LMP1), is a functional mimic of the cellular receptor CD40, but signals to B lymphocytes in an amplified and sustained manner compared with CD40. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic domain of LMP1 binds the signaling adaptor TRAF2 with lower avidity than the cytoplasmic domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. We thus tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3, and their distinct signaling characteristics. CD40 and LMP1 mutants in which the TRAF binding site sequences were swapped were examined, testing TRAF binding and degradation, and induction of B cell activation. Results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs LMP1 signaling properties. Examination of TRAF binding, degradation, cytokine production, IgM secretion, and the activation of c-Jun kinase and NF-kappaB revealed that some events are dictated by TRAF binding site sequences, others are partially regulated, and still others are independent of the TRAF binding site sequence.

The CD154-CD40-mediated costimulatory pathway is critical for T-B cell cooperation in thymus-dependent (TD) immune response in mammals. However, little is known about its existence and occurrence in lower vertebrates. Here, we report on the identification and functional characterization of CD154 and CD40 homologs from the zebrafish (Danio rerio) model. Zebrafish CD154 is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, whose tertiary structure is a sandwich containing two stacked sheets with "jelly roll" topology, just as the human TNF members do. The zebrafish CD40 is a type I membrane-bound protein with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The consensus TNFR-associated factor (TRAF)2- and TRAF6-binding motifs in mammalian CD40 are found in the cytoplasmic tail of zebrafish CD40, which indicates similar signal transduction mechanisms to higher vertebrates. Zebrafish CD154 and CD40 are widely distributed and can be up-regulated by thymus-dependent Ag. The production of IgM was dramatically decreased by anti-CD154 or soluble CD40, and it was enhanced by soluble CD154 or CD154-encoding plasmid in vivo. Thymus-dependent Ag-induced CD154 expression was inhibited by cyclosporin A, suggesting that CD154 functionally associates with T cells. Double immunofluorescence staining showed that CD40 and membrane IgM colocalized in B cells. CD154-CD40 binding assays showed that CD154 specifically binds to CD40 at homodimeric form. Our results provide the first evidence for the existence of the functional CD154-CD40-mediated costimulatory pathway and helper T cell regulatory mechanism underlying adaptive immunity in a fish species.

CD40 is a costimulatory protein expressed on the surface of many different cells. It delivers signals regulating diverse cellular responses, including proliferation, differentiation, growth suppression, and cell death. In this study, we report a novel CD40 mutant (c.234C>A or p.H78Q) that is expressed in the U266 cell line and in freshly isolated tumor cells. Three-dimensional structural model and Scatchard analysis revealed that the mutated residue located in a region is important for binding to CD40L (CD154). Functional analysis indicated that the mutated CD40 was translocated to the CD40 signalosome and involved in CD40 signal transduction. In conclusion, the mutation in CD40 can lead to an alteration of function, including the change of antigen epitope and the binding affinity with CD40L.

CD40 signaling is critical for innate and adaptive immunity against pathogens, and the cytoplasmic domain of CD40 is highly conserved both within and between species. A novel missense single nucleotide polymorphism (SNP) in the cytoplasmic domain of CD40 at position 227 (P227A) was identified, which resides on a conserved ancestral haplotype highly enriched in persons of Mexican and South American descent. Functional studies indicated that signaling via human (h) CD40-P227A stably expressed in several B-cell lines led to increased phosphorylation of c-Jun, increased secretion of the pro-inflammatory cytokines interleukin (IL)-6 and TNF-alpha, and increased Ig production, compared with wild-type hCD40. Cooperation between hCD40-P227A signaling and B-cell receptor (BCR)- or Toll-like receptor 9 (TLR9)-mediated signaling was also enhanced, resulting in elevated and synergistic production of IL-6 and Ig. We have thus identified a novel genetic variant of hCD40 with a gain-of-function immune phenotype.

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40(-/-) deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40(-/-) mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent important therapeutic modalities for neurodegenerative and neurodevelopmental disorders, as well as, for enhancement of neurogenesis.

Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.

The integral membrane protein CD40 was found on the surface of B lymphocytes that interact with CD40L on T cells during the immune response. The hydrophobic transmembrane domains of membrane proteins can be stabilized in detergent or in lipid bilayers such as liposomes. Membrane proteins can be incorporated into the liposome in a similar fashion to the way they are handled in vivo. In this study, a large amount of full-sequence CD40 was produced using a bacterial system that contained a Mistic construct. The CD40 was then reconstituted into liposomes by detergent-mediated reconstitution. All stages in the process of liposome disruption with various detergent ratios were easily observed by monitoring the optical density. The structure of the liposome and the reconstitution of CD40 were confirmed by cryo-TEM. The results of the present study show that the detergent ratio had an effect on the structure of the liposome and the amount of CD40 that was reconstituted into the liposome.

Xenotransplantation is a promising alternative to overcome donor shortage in transplantation. CD40 molecule plays an important role in the interaction of T cells with antigen-presenting cells and in the activation of vascular endothelial cells. We investigated whether the xenogeneic interaction between human CD40L (hCD40L) on T cells and porcine endothelial CD40 (pCD40) can activate porcine endothelial cells (PECs). The interaction between hCD40L and pCD40 induced the expression of chemokines on PECs as well as MHC and adhesion molecules. Furthermore, NF-kappaB signaling was activated in HEK 293 cells expressing pCD40 and PECs by stimulation with hCD40L+ Jurkat T clones. Both anti-CD40L neutralizing antibodies and NF-kappaB signal inhibitors interfered with immune activation of PECs. Overall, this study shows that xenogeneic interaction between hCD40L and pCD40 can activate PECs through NF-kappaB signaling, and therefore may contribute to acute vascular rejection in xenotransplantation.

TNF family members play crucial roles in mammalian B-cell differentiation and function, many of which have not been demonstrated in other species. To investigate the avian CD40/CD40L system, a chicken CD40 cDNA, obtained by expression screening, was used to raise monoclonal antibodies showing that CD40 was expressed on chicken B cells, monocytes and macrophages, like mammalian CD40. CD40 ligand fusion protein supported the proliferation of B cells in culture for up to 3 weeks, during which they differentiated towards a plasma cell phenotype. CD40L-activated B cells from immunised birds secreted antigen-specific IgM and IgG. These results showed important conserved functions of CD40 and its ligand in mammals and birds. CD40L provides a means for maintenance and differentiation of untransformed chicken B cells in culture, for the first time, allowing new approaches to study of post-bursal B cell biology and host-pathogen interactions with B cell tropic viruses.

Synthetic multivalent ligands, owing to the presence of multiple copies of a recognition motif attached to a central scaffold, can mediate clustering of cell surface receptors and thereby function as effector molecules. This paper dissects the relationship between structure and effector function of synthetic multivalent ligands targeting CD40, a cell surface receptor of the tumor necrosis factor receptor (TNF-R) superfamily. Triggering CD40 signaling in vivo can be used to enhance immunity against intracellular pathogens or tumors. A series of multimeric molecules has been prepared by systematically varying the shape and the valency of the central scaffold, the nature and the length of the linker as well as the sequence of the receptor binding motif. The data reported here (i) suggest that radial distribution of CD40-binding units and C3-symmetry are preferred for optimal binding to CD40 and signaling, (ii) underscore the importance of choosing an appropriate linker to connect the receptor binding motif to the central scaffold, and (iii) show the versatility of planar cyclic alpha- and beta-peptides as templates for the design of CD40L mimetics. In particular, the (Ahx)3-B trimeric scaffold-linker combination equally accommodated binding elements derived from distinct CD40L hot-spot regions including AA" loop and beta-strand E. The use of miniCD40Ls such as those reported here is complementary to other approaches (recombinant ligands, agonistic anti-receptor antibodies) and may find interesting therapeutic applications. Furthermore, the results disclosed in this paper provide the basis for future design of other TNF family member mimetics.

Increasing evidence suggests a regulatory role for leptin, an adipocyte-derived hormone, in immunity. Although recent studies indicated an essential role of leptin signaling in dendritic cell (DC) maturation, the molecular mechanisms by which leptin modulates DC functional maturation remained unclear. In this study, we showed that leptin induced CD40 expression in murine DC and significantly up-regulated their immunostimulatory function in driving T cell proliferation. Moreover, leptin markedly enhanced lipopolysaccharide-mediated DC activation. Using pharmacological inhibitors for Akt, STAT-1alpha, or NF-kappaB and the dominant negative forms of Akt and IkappaB kinase alpha/beta/gamma, as well as small interfering RNA for STAT-1alpha, we showed that Akt, STAT-1alpha, and NF-kappaB were important for the leptinor lipopolysaccharide-induced CD40 expression. Coimmunoprecipitation analysis revealed that leptin promoted immune complex formation between Akt and the IkappaB kinase subunits as well as STAT-1alpha. Blocking the activity of Akt demonstrated a crucial role for Akt in translocation of STAT-1alpha and NF-kappaB to the nucleus and activation of the CD40 promoter. Further analysis with chromatin immunoprecipitation assay confirmed that leptin recruited STAT-1alpha, NF-kappaBp65, and RNA polymerase II to the CD40 promoter and enhanced histone 4 acetylation in a time-dependent manner. Thus, our results have elucidated the molecular mechanisms underlying leptin-induced CD40 expression and DC maturation.

The recruitment of tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFkappaB signaling pathways. Moreover, while lacking germinal center formation, several hallmarks of humoral immune responses including clonal B-cell activation/expansion, antibody isotype switching, and affinity maturation remain normal. This study demonstrates a new functional domain in CD40 that controls critical aspects of B-cell immunity in an in vivo setting.

Aim: To assess the role of CD40/CD154 interaction in GO pathogenesis and to estimate usefulness of soluble CD40 (sCD40) and CD154 (sCDI54) measurements as markers of GO activity., Material and Methods: 61 individuals in 4 groups: (1) 15 euthyroid patients with clinical symptoms of ophthalmopathy (GO) who underwent corticosteroid therapy consisting of intravenous infusions of methylprednisolone (MP) and subsequent treatment with oral prednisone (P) and teleradiotherapy (TR); (2) 14 patients with hyperthyroid GD (GDtox); (3) 22 patients with GD in euthyreosis treated with methimazol (GDeu); (4) 10 healthy volunteers age and sex-matched to group 1-3. The serum samples were collected 24 hours before MP, 24 hours after MP, after TR and at the end of therapy. Serum CD40, CD154 and TPOab were determined by ELISA and TSHRab by RIA., Results: Serum concentrations of CD40 (in pg/ml) and CD154 (in ng/ml) were increased in GO patients: 84.9 (74.7-93.9) and 4.0 (2.5-7.3) respectively in comparison to controls (p < 0.001 and p < 0.05 respectively). Serum CD154 in GO group was elevated as compared to both hyperthyroid and euthyroid GD without clinical ophthalmopathy (p < 0.001 both). The sCD40/sCD154 quotient was significantly elevated during GO therapy with CS and TR in nonresponders after MP (p < 0.05) and at the end of the study (p < 0.01)., Summary: Our data suggest an important role of CD40/ CD154 interaction in the pathogenesis of autoimmune process leading to inflammatory infiltration in Graves' ophthalmopathy, however usefulness of sCD40 and sCD154 measurements in prediction of effects of GO treatment and its monitoring needs further investigations.

CD40 is a member of the growing tumor necrosis factor receptor (TNF-R) family of molecules, and has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154 expressed on activated T cells stimulates B cell proliferation, differentiation, isotype switching, upregulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. The present review will summarize recent literature data on the various CD40 signalling pathways, which involve both the TNF-R associated factors (TRAFs) and additional signalling proteins, and lead to activation of kinases and transcription factors.

Aim: To measure the level of serum soluble CD40 (sCD40) in patients with acute hepatitis, hepatitis gravis and primary carcinoma of the liver, and to evaluate the relationship of sCD40 with biochemical marks and disease prognosis., Methods: Patients with acute hepatitis (n=49) hepatitis gravis (n=22) and primary carcinoma of the liver (n=13) were studied, and serum sCD40 was determined in these patients and compared with that of healthy controls (n=44) by enzyme linked immunosorbent assay (ELISA). The binding capacity of serum sCD40 to its ligand CD40L was detected by flow cytometry (FCM) in vitro., Results: Concentration of sCD40 was significantly higher in patients with liver disease than that in healthy controls (P<0.001), but no significant difference was found between the three types of liver disease (P=0.475). In the hepatitis gravis group, sCD40 concentration in dead patients was higher compared with that in the survivals (P<0.05). Level of sCD40 in patients with acute hepatitis was correlated with serum alanine transaminase (ALT) and aspartic transaminase (AST). The serum sCD40 could bind CD40L in vitro., Conclusion: These data suggest that sCD40 is an important serological marker in liver disease to evaluate acute injury of hepatocytes, and it shows a relevance with the prognosis of hepatitis gravis. The highly elevated level of sCD40 suggest the involvement of CD40 and its ligand CD40L in liver disease.

Aim: To investigate the effect of incorporation of an isoleucine zipper (IZ) motif into CD40 on binding activity of CD40 for the CD40 ligand (CD40L)., Methods: Prokaryotic expression vectors for 2 soluble CD40 derivatives, shCD40His and shCD40IZ containing an IZ domain, were constructed and expressed in Escherichia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their binding activity for CD40L on Jurkat T cells was determined by flow cytometry., Results: shCD40His and shCD40IZ were generated. Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface. shCD40IZ had much higher binding activity to its ligand (CD40L) than did shCD40His. Furthermore, size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution., Conclusion: Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.